PROJECT ABSTRACT Rheumatoid arthritis (RA) is a common systemic autoimmune disease that causes joint deformity, bone erosion, and subchondral and systemic bone loss, resulting in disability at 4 years after onset as high as 31%. Bone damage in this disease is driven by increased osteoclast (OC)-mediated bone resorption and decreased osteo- blast (OB)-mediated bone formation. Our preliminary data points to an important role for B cells in RA with the presence of activated B cells in rarely studied target tissue such as the synovium, subchondral bone, and bone marrow where OBs and OCs develop. Based on in vitro studies, these abnormal B cells can produce cytokines such as RANKL and TNF that mediate OC activation and OB dysfunction. As a novel approach to dissect in situ cell function in RA target tissue, we have recently begun to develop techniques for laser capture micro-dissection (LCM) of synovium and bone. Despite the great promise of such an approach, single cell RNAseq on bone tis- sue remains a technical challenge. A major focus of the current application is to establish rigorous and reproduc- ible LCM protocols as a prerequisite for the desired transcriptomic studies critical to move the field forward. The approaches developed here will allow in situ interrogation of the role of B cells in bone homeostasis. To sur- mount technical barriers, we plan to leverage a unique collaboration between University of Rochester (UR) Aller- gy, Immunology, and Rheumatology (AIR), UR Orthopedics, and the UR Accelerating Medicines Partnership (AMP) NIH Network with access to surgical and biopsy samples including from untreated RA patients and state- of-the-art technologies within the Center for Musculoskeletal Research (CMSR) and UR. We will optimize and validate LCM protocols for synovium and bone in order to begin to define cell function within the 3D joint milieu via 2 specific aims: 1: Optimize LCM protocols to define cell-cell interactions in the synovial and bone microenvi- ronment; 2: Investigate the mechanisms of B cell driven OC activation and OB inhibition in RA target tissue. In Aim 1 we will establish robust and reproducible methods in control tonsil tissue and RA arthroplasty samples by optimizing the following parameters: 1) Tissue preparation and staining to identify cells of interest and interacting populations- fixation method/timing, antibody combinations/timing; 2) Cell capture- numbers of cells, timing; 3) RNA isolation and library preparation. In Aim 2 we will validate the utility of our approach by defining discrete B cell subset function and asking how B cells in close proximity to OC and OB precursors impact the bone lineage cell populations using RNA sequencing and pathway analyses of single cell sorted compared to laser captured cells. The approaches developed in this R21 will allow synovium and bone research to move in a new direction by focusing on in situ cell function, in the process defining unexplored and key mechanisms that underlie aber- rant B cell interactions with both OCs and OBs and mediate joint destruction in the target tissue.